The adenovirus 2 major late promoter (MLP) is among the most active promoters transcribed in vitro using nuclear extracts from uninfected cells. Mutational analysis of the Ad2 MLP previously determined that sequences at and proximal to the TATA box (-31 to -25) were essential for a basal level of transcriptional activity. Basal activity could be increased 10-20 fold by binding of a trans-acting factor to an upstream promoter sequence. The two factors binding to the TATA and UPS, TFIID and MLTF respectively, appear to bind cooperatively to the Ad2 MLP. The region of the Ad2 MLP surrounding the CAP or transcription start site (+1) has not been as well characterized. However, mutation of sequences at and downstream of the CAP site lowers the efficiency of transcription. These studies suggest, therefore that the sequences at and surrounding the CAP site might define a cis-acting regulatory sequence required for accurate regulation of transcription. In this study, the protein DNA interactions at the Ad2 MLP CAP site were investigated by DNase I footprint analysis and mobility gel shift analysis. These studies showed that distinct polypeptide factors bind to the TATA box and CAP site sequences. Mutation of the CAP sequence does not affect the interaction of TFIID with the TATA box nor is binding of the factor recognizing the CAP site affected by depletion of TFIID. When the CAP sequence is mutated, transcriptional activity of the Ad2 MLP is reduced both in vitro and in vivo. Depletion of nuclear extracts of CAP binding activity also reduces Ad2 MLP transcriptional activity. DNA affinity-purified CAP binding factor, when added back to the depleted extract, is able to restore activity to control levels. TFIID-CBF complexes can also be demonstrated prior to binding to the AD2 MLP. Efforts are currently underway to purify the CAP site binding factor to homogeneity and obtain a cDNA clone. Expression of the factor will then allow biochemical studies to define its mechanism of action during establishment of the transcription complex at the Ad2 MLP as well as host cell genes.